Real time identification of analytes in a complex biological fluid is difficult, and requires careful thought as to (a) the preparation of the sample, (b) whether a separation step is required to simplify the signal, (c) whether a detection method can be employed which has no effect on the sample itself, (e.g. non-destructive). An ideal device would allow rapid detection of a wide range of simple or complex molecules in the liquid phase, at biological concentrations, and yield information about chemical structure and composition. A desirable feature of the detection method would be to enable the separation criteria to be relaxed such that sample preparation and detection could occur in series, without the need for complex separation technology. An on-line detector is particularly advantageous when sample size is limited, and additional analysis of the sample is required. Moreover, mass spectrometry ("MS") and NMR are detection methods well suited to yielding high quality chemical information for multi-component samples, requiring no a priori knowledge of the constituents.
Though much has been discussed in the literature towards realizing integrated separation technology including sample preparation and separation devices, and associated fluidics so that low yield or precious samples may be prepared and analyzed, little has been realized to date. In sample analysis instrumentation, particularly in separation systems involving capillary electrophoresis or liquid chromatography, smaller dimensions of the sample handling conduits and separation compartments result in improved performance characteristics, while reducing cost of production and analysis. Miniaturization of the sample preparation or separation region, to result in small sample volume requirements, necessarily means a greater demand on the detection method both by virtue of sample volume and potentially, sensitivity.
There are many types of detection methods possible. Optical transmission methods such as refractive index, ultraviolet-visible ("UV-VIS") and infrared ("IR") are relatively inexpensive, but are unable to give complex chemical structure and composition information. Furthermore they are path-length limited and sensitivity of detection is limited. Infrared spectroscopy is relatively insensitive, particularly to contaminants, and yields only functional group or fingerprint identification. MS is a sensitive method giving mass information; however, MS has the drawback of requiring sample preparation for nonvolatile analytes, as well as being destructive to the sample.
One of the most powerful analytical methods for molecular structure information is NMR. NMR provides spectral information as a function of the electronic environment of the molecule and is nondestructive to the sample. In addition, reaction rates, coupling constants, bond-lengths, and two- and three-dimensional structure can be obtained with this technique. The strength of both NMR and MS is the ability to derive fundamental chemical structure information, which is high resolution in terms of either chemical shift or mass, yielding the possibility of simultaneous analysis of multiple species. The inherent insensitivity of the NMR method however, has limited its usefulness as a detection method for liquid phase analysis of very small samples, such as effluent from a liquid chromatography or capillary electrophoretic separation.
NMR combined with liquid chromatography or capillary electrophoresis was demonstrated as early as 1978 using stopped flow (Watanabe et al. (1978) Proc. Jpn. Acad. 54:194), and in 1979 with continuous flow (Bayer et al. (1979) J. Chromatog. 186:497-507), though limitations due to solvent as well as inherent sensitivity curtailed the use of the method. See Dorn et al. (1984) Anal. Chem. 56:747-758 for a review.
Recent experiments using NMR as a detector for nanoliter sample volumes, suggests that NMR could provide a greater detection sensitivity than in previous investigations. Wu et al. (1994a) J. Am. Chem. Soc. 116:7929-7930; Olson et al. (1995) Science 270:1967-1970; Wu et al. (1994b) Anal. Chem. 66:3849-3857; and Wu et al. (1995) Anal. Chem. 67:3101-3107. Unfortunately, though observations were made on nanoliter volumes, the findings translate into millimolar levels of detection sensitivity.
A number of areas can be targeted to increase the sensitivity of NMR detection for liquid phase analysis. Resistive losses, operating temperature, sample ionic strength, filling factor, and coil geometry affect the sensitivity of the coil. Cooling the radiofrequency coil and using superconducting coil material have resulted in some gain in signal-to-noise through reduction in coil resistance and thermal properties. However, it is difficult to achieve the theoretical maximum, since detecting signal from a room temperature liquid sample using a cryogenically cooled radiofrequency probe has proven difficult.
The NMR signal-to-noise is directly proportional to the sample volume (V.sub.s) interrogated by the detection coil (filling factor), the magnetization per unit volume (M.sub.o), and the strength of the radiofrequency ("RF") field (B.sub.1) per unit current, and inversely proportional to the square root of the coil resistance (R): EQU Signal.varies.(V.sub.s.times.M.sub.o.times.B.sub.1)/R
Signal-to-noise can be maximized by decreasing the coil radius, and matching the coil inner diameter as close to the size of the sample as possible. Inadequate filling factor will generally be an issue when standard radiofrequency NMR coils are used to detect signal from very small sample volumes, e.g., from a microcolumn or other miniaturized sample preparation technology. Reduction in the size of NMR radiofrequency coils to the diameter of the fused glass capillary used for these types of separations, has allowed detection of signal from nanoliter volumes from on-line capillary electrophoretic separations Wu et al. (1994a), supra; Olson et al. (1995), supra; Wu et al. (1994b), supra; Wu et al. (1995), supra.
A solenoid microcoil detection cell formed from a fused silica capillary wrapped with copper wire has been used for static measurements of sucrose, arginine and other simple compounds. Wu et al. (1994a), supra; Olson et al. (1995), supra. Coil diameter has been further reduced by the use of conventional micro-electronic techniques in which planar gold or aluminum R.F. coils having a diameter ranging from 10-200 .mu.m were etched in silicon dioxide using standard photolithography. Peck (1995) J. Magn. Reson. 108(B) 114-124. The signal-to-noise ratio (SNR) of these planar micro-coils for analyzing solid samples, e.g., silicon rubber was increased by a factor of 10 over other coils. For significant advancement in hyphenating NMR with LC or CE methods, an approach allowing micromolar or even nanomolar limits of detection is required however.
Factors affecting the limit of detection can also be attributed to bulk susceptibility shifts, which become dominant when the sample volume is of the order of the size of the sample chamber and coil. This is in addition to the coil geometry, resistive losses, sample ionic strength, filling factor and operating temperature considerations previously mentioned. We have constructed susceptibility-matched microcoils using 50 .mu.m copper wire with an inner diameter of 70 .mu.m, and obtained signal in 64 seconds from a 12.5 mM solution of arginine at 400 MHZ, with a signal-to-noise of 6:1. Or, in other words, normalizing these results to 300 MHZ for direct comparison with U.S. Pat. No. 5,654,636, issued Aug. 5, 1997, to Sweedler et al. this yields a signal-to-noise of 3:1 versus 1:1 obtained in Sweedler et al.
In order to obtain signal from nanoliter-volume samples having analyte in the micromolar concentration range, assuming limitations only of currently available field strength (750 MHZ) and a time constraint of observing signal after one minute of acquisition time from 5.4 nl of volume, the following parameters could be optimized: (a) reduce the wall thickness while keeping the sample volume the same increasing the filling factor; (b) increase the "Q" or quality factor of the coil by using a superconducting coil; and/or (c) employ sensitivity enhancement techniques such as decoupling or Nuclear Overhauser Enhancement (NOE), or optical pumping (with .sup.3 He). The following table provides a comparison of the limitation-of-detection before and after implementation of the above mentioned factors. The numbers below were obtained by converting the theoretical S/N advantage obtained by each method to time saved using the method. One minute was deemed acceptable; 1 hour is shown for comparison purposes.
Acquisition Duration Limit of Detection of Analyte (mM) 1 minute (before optimization) 4.2 1 minute (after optimization) 8.5 .times. 10.sup.-3 1 hour (after optimization) 1.05 .times. 10.sup.-3
These calculations indicate that an integrated system could be constructed with the ability to detect micromolar quantities of analyte contained in nanoliter samples using in-line NMR detection following sample preparation and separation.
While silicon micro-machining has been useful in the fabrication of miniaturized liquid phase analysis systems, improvements have been made to overcome the inherent shortcomings of this technique. For example, U.S. Pat. No. 5,500,071 to Kaltenbach et al., U.S. Pat. No. 5,571,410 to Swedberg et al and U.S. patent application Ser. No. 08/656,281 to Kaltenbach et al., disclose the use of laser ablation to form microstructures in novel polymer substrates. This permits an enhanced symmetry and alignment of structures formed by component parts, enhanced separation capabilities, avoidance of problems with SiO.sub.2 chemistry, low-cost manufacturing, the formation of microstructures of any size and geometry, and the incorporation of a detection means for on-column analysis. The advantage of combining miniaturized planar liquid phase analysis systems with on-column NMR detection is clearly advantageous in terms of lower overhead for instrument maintenance, increased speed of analysis, decreased sample and solvent consumption, full automation capabilities, increased detection efficiency, and increased quality of information.
A method and an apparatus for NMR spectroscopy of samples from online separation methods has been described. Sweedler et al., supra. While the problems of susceptibility and signal-to-noise from samples of an online separation apparatus are addressed therein, the method and apparatus described is limited to analysis of simple aqueous solutions. The apparatus includes a capillary channel etched or grooved in a substrate such as glass or polycarbonate and a planar lithographic microcoil. The use of micron-feature devices with integrated sample preparation and detection is not described. Integration of the NMR coil with the separation device eliminates dead volume which increases the dispersion and drastically degrades resolution between the point of chemical separation and detection. The method and apparatus presented in Sweedler et al. uses a conventional NMR spectrometer, such that sample preparation occurs outside of the separation/detection system, and hence a truly integrated solution for sample preparation and detection is lacking.
Accordingly, there is a need in the art to address the current trend to move away from expensive instrumentation in a central lab setting, to a low cost, portable, fast time-to-result hyphenated system, requiring little user knowledge to analyze complex samples. Examples of complex samples include chemical or biochemical species in complex biological matrices, or chemical species in complex samples such as soil, sea water, waste water, sludge found at remediation sites, and the like.
There is yet a need in the art for a miniaturized device that avoids the problems of chemical and pH instability that are typical with SiO.sub.2 substrates and has miniaturized liquid sample handling capabilities and on-board sample preparation and NMR detection means.